Western blot analysis of p42-ERK and phospho-ERK1/2 expression was performed by loading 50 µg HeLa (lane 1 and 2, HeLa (human), NRK (lane 3 and 4, NRK (rat) and MDCK (lane 5 and 6, MDCK (canine) cell lysates with or without TPA treatment onto a 10% Tris-Glycine polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane and blocked with 1% BSA/TBST for at least 1 hour at room temperature. p42 ERK was detected using a mouse monoclonal antibody (Product # MA1-099) and phospho-ERK1/2 was detected using a rabbit monoclonal antibody (Product # 700012) at a concentration of 1 µg/mL in blocking buffer overnight at 4°C on a rocking platform. Blots were then incubated with a goat anti-mouse IgG Alexa Fluor790 secondary antibody (Product # A11357, green, top panel) and a goat anti-rabbit IgG Alexa Fluor 680 secondary antibody (Product # A-21109, red, bottom panel) at a dilution of 1:10,000 for at least 1 hour. Fluorescent detection was performed using the Odyssey® CLx imaging system (Li-cor Biosciences). Images generated by Joell Solan in Paul Lampe Lab at Fred Hutchinson Cancer Research Center.